Molecular chaperones and their respective co-chaperones make up an important class of protein molecules found in the cells of all organisms. They are found in the major cellular compartments within eukaryotic cells. They participate in a variety of cellular processes such as folding of newly formed polypeptides, assembly and disassembly of multimeric protein structures, protein translocation across membranes, and protein degradation. This research proposal takes a molecular and biochemical approach to the structure-function analysis of the molecular cochaperone GrpE protein from E. coli. The specific aims are to begin to identify the structure of the GrpE protein and then determine how the structure relates to the known function of GrpE, which is to assist the DnaK chaperone in its various duties. A number of different approaches will be utilized. Specific deletion mutant proteins will be constructed based on stable protein fragments that are generated from digestion of the full-length protein with a nonspecific protease and additionally, mutants will be designed from information about highly conserved regions of the grpE gene family. These deletion mutant proteins will then be subject to a variety of different functional assays to see if certain activities are lost or altered in any way. Conditions for crystal growth will be explored for the GrpE protein and any interesting deletion mutants in order to obtain detailed x-ray crystallographic structural information. Crosslinking studies will be carried out to determine the site or sites of interaction between the GrpE protein and the DnaK protein. Conditions for crystal growth of a crosslinked GrpE-DnaK complex will also be explored. Finally, the beginnings of a biophysical study are proposed to investigate the dynamics of the GrpE-DnaK interaction through the design and synthesis of molecules that will contain a highly conserved amino acid loop structure that will mimic that which is found in the native DnaK protein. Results from this study may help to unravel the intricacies of this unique protein-protein interaction and provide a better understanding of how large biological molecules communicate with each other.